Throughout this application various publications are referenced, many in parenthesis. Full citations for each of these publications are provided at the end of the Detailed Description. The disclosures of each of these publications in their entireties are hereby incorporated by reference in this application.
After injury, a fibrin-rich clot fills the wound as a result of the local extravasation of plasma (Clark 1993b). This fibrin-rich provisional matrix, which also contains other matrix components like fibronectin (FN) and vitronectin as well as fibrin (Clark et al. 1981), forms a scaffold for the inward migration of the cells involved in early wound repair (Clark 1993a; Clark 1993b; Clark et al. 1982a; Clark 1996; Clark et al. 1996). Concomitantly, platelets release a plethora of growth factors, some of which bind to the fibrin meshwork. Subsequently, blood leukocytes, especially neutrophils and monocytes, migrate into the fibrin-rich provisional matrix. The neutrophils phagocytize and kill contaminating microorganisms (Tonnesen et al. 1988), while the monocytes mature into growth factor-producing macrophages (Shaw et al. 1990). Fibrin undergoes intermolecular crosslinking by formation of .epsilon.-(.gamma.-glutamyl)lysine .epsilon.-(.gamma.-glu)lys! isopeptide bonds in the presence of plasma transglutaminase (Pisano et al. 1968). Crosslinking of .gamma. chains within fibrils forms dimers while intermolecular crosslinking among .alpha. chains creates oligomers and larger chain polymers (Mosesson et al. 1989; Shainoff et al. 1991). More recent in vitro studies indicate that in addition to .gamma. dimers, higher order forms of crosslinked .gamma. chain multimers form slowly and progressively over a period of hours to days (Siebenlist and Mosesson 1992). Coagulation factor XIIIa stabilizes the structure of the provisional matrix by cross-linking fibrin homocomplexes (Lorand 1972), and fibrin and FN heterocomplexes (Mosher 1975; Mosher 1976). FN is probably an important part of the invasion process in vivo since it is able to bind to cells and to other extracellular matrix (ECM) proteins simultaneously, and since fibroblasts can use FN as a substrate for migration in vitro (Hsieh and Chen 1983). Simultaneous with clot evolution during the first three days after injury, fibroblasts and endothelial cells in the underlying subcutaneous tissue proliferate (Clark 1993b). Fibroblasts and endothelial cells appear most numerous along the edge and base of the wound, with lesser numbers further away. By the third day after injury increased numbers of fibroblasts greatly expand the subcutaneous fibrous septae coursing between the fat lobules beneath the wound, and envelop individual adipocytes in proximity to the wound as well. Nevertheless, no fibroblast invasion of the wound clot is observed. Endothelial cells within blood vessels adjacent to the wound proliferate, causing marked vessel hypertrophy but do not move into the wound space (Clark et al. 1982b; Clark et al. 1982c).
Three days after injury, fibroblasts expressing abundant provisional matrix integrins (Xu and Clark 1996) migrate from the collagenous matrix of the dermis into the wound matrix as part of granulation tissue formation. Presumably this transmigration is in response to platelet-derived growth factor (PDGF) (Seppa et al. 1982; Senior et al. 1983) and other growth factors released by platelets and monocytes (Shimokado et al. 1985; Ross et al. 1986; Rappolee et al. 1988). Although PDGF is a potent mitogen and chemoattractant for fibroblasts (Seppa et al. 1982; Ross and Raines 1990), the full set of functional requirements for fibroblast transmigration from one matrix into another have not been defined.
Due to the complexity of the in vivo situation, three different in vitro models have been used in the past to study fibroblast invasion of a fibrin clot. Graham et al. (Graham et al. 1984) embedded explants of chick flexor tendons in a fibrin matrix and studied the migration, proliferation, and collagen synthesis of the fibroblasts. Migration in this system was induced by fetal bovine serum (FBS) but not by physiological concentrations of platelet lysate or PDGF. Brown and his coworkers (Brown et al. 1993) overlaid fibroblasts attached to tissue culture dishes with a thin fibrin layer and observed that cells migrated into the fibrin within 24 hours. This migration process was dependent on the nature of the fibrin clot: cross-linking of the fibrin .alpha.-chains by factor XIIIa enhanced the number of invading cells. Knox et al. observed that the presence of FN (Knox et al. 1986) and plasminogen and its activators (Knox et al. 1987) were necessary for fibroblast invasion of a plasma clot on which the fibroblasts were seeded.
Before invasion of the fibrin clot, however, and in contrast to these in vitro models, resident tissue fibroblasts in normal dermis are surrounded by a matrix mainly composed of type I collagen. An often used in vitro model for the dermis is a relaxed hydrated collagen gel with embedded fibroblasts which acquire the dermal phenotypic characteristics of resident dermal fibroblasts (Elsdale and Bard 1972; Bell et al. 1979; Grinnell 1994). Just as in vivo, there is a low level of cell proliferation (Sarber et al. 1981) and collagen biosynthesis (Mauch et al. 1988), but an increased release of collagenase (Nusgens et al. 1984; Unemori and Werb 1986). Furthermore, fibroblasts in relaxed collagen gels are less responsive to growth factors (Nakagawa et al. 1989; Nishiyama et al. 1990). An explanation for the different behavior of cells in the collagen gel in comparison to tissue culture plastic is that different kinds of extracellular matrix can dramatically affect cell functions and behavior by regulating gene expression and second messenger pathways (Hay 1991; Clark et al. 1995; Streuli et al. 1995; Tremble et al. 1995; Xu and Clark 1996). For example, when fibroblasts are cultured in collagen gels, .alpha.2 integrin gene expression is increased (Klein et al. 1991; Xu and Clark 1996) and the autophosphorylation of PDGF-receptor is decreased (Lin and Grinnell 1993).
Integrins are a family of cell surface receptors which are primarily responsible for cell adhesion (Hynes 1992). All integrins are composed of one .alpha. and one .beta. subunit. A large number of integrins are responsible for the interaction of fibroblasts with the proteins of the provisional matrix. The integrins .alpha.3.beta.1, .alpha.4.beta.1, .alpha.5.beta.1 and .alpha.v.beta.3 mediate adhesion of adult human dermal fibroblasts to FN (Elices et al. 1991; Gailit et al. 1993; Gailit and Clark 1996). .alpha.v.beta.3 is the only known fibroblast integrin that recognizes fibrinogen (Smith et al. 1990; Gailit and Clark 1996). When wound fibroblasts migrate into the fibrin/FN-rich clot, .alpha.3.beta.1 and .alpha.5.beta.1 fibronectin receptors, but not .alpha.2.beta.1 collagen receptors, are dramatically upregulated (Xu and Clark 1996). .alpha.2.beta.1 collagen receptors increase on wound fibroblasts later at day 7 when collagen accumulates in the wound area (Welch et al. 1990; Clark et al. 1995).
A need continues to exist for an understanding of the regulation of interactions between cells and extracellular matrix and the functional mechanisms involved during wound repair.